The present invention relates to a method for producing an L-amino acid by fermentation at high industrial efficiency.
As a direct fermentation method for producing and accumulating an L-amino acids directly from saccahride, there have been known methods in which mutant strains derived from wild-type strains of microorganism belonging to the genus Corynebacterium, Brevibacterium, Escherichia, Serratia or Arthrobacter are employed For example, the following are known as L-amino acid-producing mutants: auxotrophic mutants which require amino acids, etc. (Japanese Published Examined Patent Application No. 10037/1981), mutants which have resistance to amino acid analogs and vitamins (Japanese Published Unexamined Patent Application Nos. 134993/1981 and 44193/1987), mutants which have both auxotrophic mutation and resistance mutation to amino acid analog(Japanese Published Unexamined Patent Application Nos. 31093/1975 and 134993/1981), mutants which have lowered degradability (Japanese Published Unexamined Patent Application No. 273487/1988, and Japanese Published Examined Patent Application No. 48195/1977), and mutants whose aminoacyl t-RNA-synthesizing enzymes have a decreased substrate affinity (Japanese Published Unexamined Patent Application No. 330275/1992).
It has also been known that the production of an amino acid can be improved by using transformants obtained by transformation with recombinant DNAs carrying genes involved in the biosynthesis of amino acids (Japanese Published Unexamined Patent Application Nos. 893/1983, 12995/1985, 210994/1985, 30693/1985, 195695/1986, 271981/1986, 458/1990 and 42988/1990; Japanese Published Examined Patent Application s. 42676/1989, 11960/1993 and 26467/1993).
For producing L-tryptophan, there has been a report that the productivity of the amino acid was improved by giving resistance to aminoquinoline derivatives or to phenothiazine derivatives (Japanese Published Unexamined Patent Application No. 112795/1992).
There have been a report that the expression of an operon involved in histidine synthesis is increased in a DNA gyrase-deficient strain [Proc. Natl. Acad. Sci. USA, 84, 517 (1987)] and a report that the levels of some amino acid t-RNA species including His-tRNA are decreased in a DNA gyrase mutant strain [J. Mol. Biol., 66, 131 (1972)], however, no report has been made yet about the relation between resistance to DNA gyrase inhibitors and amino acid productivity.
An object of the present invention is to provide an industrially efficient method for producing an L-amino acid useful as medicament, chemical agent, food material and feed additive.
The present invention relates to the following aspects (1) to (14).
(1) A method for producing an L-amino acid, which comprises:
(a) culturing in a medium a microorganism having an ability to produce an L-amino acid and having resistance to a DNA gyrase inhibitor;
(b) producing and accumulating the L-amino acid in the culture; and
(c) recovering the L-amino acid from the culture.
(2) The method for producing an L-amino acid as described above in (1), wherein the DNA gyrase inhibitor is selected from the group consisting of nalidixic acid, oxolinic acid, coumermycin, novobiocin and the alkali metal salts of these substances.
(3) The method for producing an L-amino acid as described above in (1), wherein the microorganism has resistance to an aminoquinoline derivative.
(4) The method for producing an L-amino acid as described above in (3), wherein the aminoquinoline derivative is selected from the group consisting of chloroquine, amodiaquine, pentaquine, primaquine and the alkali metal salts of these substances.
(5) The method for producing an L-amino acid as described above in any one of (1) to (4), wherein the L-amino acid is L-histidine.
(6) The method for producing an L-amino acid as described above in (1) or (3), wherein the microorganism is selected from the group consisting of genera Serratia, Corynebacterium, Arthrobacter, Microbacterium, Bacillus and Escherichia.
(7) The method for producing an L-amino acid as described above in (6), wherein the microorganism is selected from the group consisting of Escherichia coli H-9342 (FERM BP-6675) and Escherichia coli H-9343 (FERM BP-6676).
(8) A microorganism having an ability to produce an L-amino acid and having resistance to a DNA gyrase inhibitor.
(9) The microorganism described above in (8), wherein the DNA gyrase inhibitor is selected from the group consisting of nalidixic acid, oxolinic acid, coumermycin, novobiocin, and the alkali metal salts of these substances.
(10) The microorganism described above in (8) or (9), wherein the microorganism has resistance to an aminoquinoline derivative.
(11) The microorganism described above in (10), wherein the aminoquinoline derivative is selected from the group consisting of chloroquine, amodiaquine, pentaquine, primaquine, and the alkali metal salts of these substances.
(12) The microorganism described above in (8), wherein the L-amino acid is L-histidine.
(13) The microorganism described above in any one of (8) to (12), wherein the microorganism is selected from the group consisting of genera Serratia, Corynebacterium, Arthrobacter, Microbacterium, Bacillus, and Escherichia.
(14) A microorganism selected from either Escherichia coli H-9342 (FERM BP-6675) or Escherichia coli H-9343 (FERM BP-6676).
As the microorganism of the present invention, any microorganism can be used, so long as it has an ability to produce an L-amino acid and has resistance to the DNA gyrase inhibitor. Additionally, it is preferable that the microorganism has further resistance to an aminoquinoline derivative. Examples of the microorganism include microorganisms belonging to the genus Serratia, Corynebacterium, Arthrobacter, Microbacterium, Bacillus, or Escherichia, such as Serratia ficaria, Serratia fonticola, Serratia liquiefaciens, Serratia marcescens, Corynebacterium glutamicum, Corynebacterium mycetoides, Corynebacterium variabilis, Corynebacterium ammoniagenes, Arthrobacter crystallopoietes, Arthrobacter duodecadis, Arthrobacter ramosus, Arthrobacter sulfureus, Arthrobacter aurescens, Arthrobacter citreus, Arthrobacter globiformis, Microbacterium ammoniaphilum, Bacillus subtilis, Bacillus amyloliquefacines and Escherichia coli. 
As the DNA gyrase inhibitor for use in the present invention, any substance can be used, so long as it inhibits DNA gyrase, one of the type II topoisomerases which are present in bacteria. For example, nalidixic acid, oxolinic acid, coumermycin and novobiocin can be used as the DNA gyrase inhibitor. Additionally, the alkali metal salts of these substances can be used as the DNA gyrase inhibitor. Herein, any alkali metal such as sodium and potassium can be used as the alkali metals.
As the aminoquinoline derivative for use in the present invention, any substance can be used, so long as it has the aminoquinoline skeleton. For example, 4-aminoquinoline derivatives such as chloroquine and amodiaquine and 8-aminoquinoline derivatives such as pentaquine and primaquine can be used as the aminoquinoline derivative. Additionally, the alkali metal salts of these substances can be used as the aminoquinoline derivative. All of these substances are known as antimalarial drugs. Herein, any alkali metal such as sodium and potassium can be used as the alkali metals.
The microorganism of the present invention can be obtained by subjecting a microorganism having an ability to produce an L-amino acid to a conventional mutation treatment including ultraviolet irradiation and the treatment with mutagen such as N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine (NTG), culturing the resulting mutant strains under general conditions on an agar plate medium containing a DNA gyrase inhibitor at a concentration at which the parent strain cannot grow or grow poorly, and selecting colonies which grow more rapidly than that of the parent strain or colonies which are larger than the parent strain among the resulting colonies.
Further, the microorganism having both resistance to a DNA gyrase inhibitor and resistance to an aminoquinoline derivative can be obtained by subjecting the DNA gyrase inhibitor-resistant strain to a mutation treatment, culturing the resulting mutant strains on an agar plate medium containing an aminoquinoline derivative at a concentration at which the parent strain cannot grow or grows poorly, and selecting colonies which are larger than the parent strain among the resulting colonies.
As the microorganism having an ability to produce the amino acid, a microorganism inherently having an ability to produce the amino acid can be used; alternatively, a microorganism which is newly obtained by subjecting a wild-type of a microorganism to produce the amino acid by known methods can also be used.
The known methods include cell fusion method, transduction method, and other gene recombinant techniques [for all, see Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (abbreviated as Molecular Cloning, 2nd ed. hereinbelow)], in addition to the above mutation treatment.
The microorganism of the present invention can also be obtained, by preparing a microorganism having resistance to a DNA gyrase inhibitor or a microorganism having both resistance to a DNA gyrase inhibitor and resistance to an aminoquinoline derivative by conventional mutation treatment and then by subjecting the prepared mutant microorganism to the above-described method to confer on the microorganism an ability to produce an L-amino acid.
Specific examples of the microorganisms of the present invention include Escherichia coli H-9342 (FERM BP-6675) and Escherichia coli H-9343 (FERM BP-6676).
The production of the L-amino acid by using the microorganism of the present invention can be carried out by an conventional method for culturing bacteria.
As the medium used for the production of L-amino acid, any of medium may be used, so long as it appropriately contains a carbon source, a nitrogen source, an inorganic substance and trace amounts of nutrients which the strain requires.
As the carbon source, carbohydrates such as glucose, fructose, lactose, molasses, cellulose hydrolysates, crude saccharide hydrolysates and starch hydrolysates; organic acids such as pyruvic acid, acetic acid, fumaric acid, malic acid and lactic acid; and alcohol such as glycerin and ethanol can be used.
As the nitrogen source, ammonia; various inorganic salts such as ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate; ammonium salts of organic acids; amines; peptone, meat extract, corn steep liquor, casein hydrolysates, soybean cake hydrolysates, various fermented cells and digested matters thereof can be used.
As the inorganic substance, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, magnesium chloride, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium chloride and calcium carbonate can be used.
The microorganism is cultured under aerobic conditions such as shaking culture and aerated agitation culture, at a temperature within a range of 20 to 40xc2x0 C., preferably within a range of 28 to 37xc2x0 C. The pH of the medium is within a range of 5 to 9, preferably around neutrality. The pH of the medium is adjusted by using calcium carbonate, inorganic or organic acids, alkali solutions, ammonia and pH buffers. Generally, the L-amino acid is produced and accumulated in the medium by culturing for 1 to 7 days.
After completion of the culturing, the precipitates such as cells are removed from the culture, and the L-amino acid can be recovered from the culture by means of ion exchange treatment method, concentration, etc., in combination.
In accordance with the present invention, any L-amino acid can be produced with no specific limitation, but includes for example L-histidine.